World Journal of Chemical Education, 2019, Vol. 7, No.

2, 45-52
Available online at http://pubs.sciepub.com/wjce/7/2/3
Published by Science and Education Publishing
DOI:10.12691/wjce-7-2-3

The Iodine Test for Reducing Sugars – A Safe, Quick

and Easy Alternative to Copper(II) and
Silver(I) Based Reagents
Holger Fleischer*

Scheffold-Gymnasium, Schwaebisch Gmuend, Germany

*Corresponding author: fleischer@seminar-stuttgart.de

Received January 17, 2019; Revised February 26, 2019; Accepted March 10, 2019
Abstract The reaction of Fehling’s and Benedicts’s test solutions with solutions of reducing sugars, e.g. glucose,
is frequently misinterpreted with respect to the products formed. The tests are by no means suitable to detect
aldehyde groups in organic molecules. The course of the reaction is known since long to be rather complex and the
corresponding carboxylate, often assumed to be the major reaction product, does not occur at all. Both tests, as well
as Tollens’ test using alkaline silver(I) solution, have some drawbacks for chemistry lessons, especially in students’
lab courses. The “iodine test”, well established to quantitatively determine glucose, could be a suitable alternative in
many cases. It allows a quick detection, even of small amounts or reducing sugars at room temperature by
decolourisation of a weakly alkaline iodine–starch–solution. The test is based on several coupled equilibria, and
hypoiodous acid is the oxidizing agent. The complexity of the molecular description of the reaction can easily be
reduced. Hence, the iodine test is suited to set different levels of difficulty, in lab work as well as in exams. The
“iodine test” can even be used in Primary School, since the necessary solutions do not represent a danger.
Keywords: iodine test, Fehling’s test, Benedict’s test, reducing sugars
Cite This Article: Holger Fleischer, “The Iodine Test for Reducing Sugars – A Safe, Quick and Easy
Alternative to Copper(II) and Silver(I) Based Reagents.” World Journal of Chemical Education, vol. 7, no. 2
(2019): 45-52. doi: 10.12691/wjce-7-2-3.

undergraduate chemistry lessons, reactivity and molecular

1. Introduction
Sugars, i.e. carbohydrates of relatively low molecular
weight, are nourishing contents of quite a lot of foods.
They typically have a sweet taste which is highly regarded
by many consumers. Thus, sugars are not only found in
foods where they naturally occur. Food producers can add
a variety of sugar sources to their products in order to give
them a sweet taste. Drawbacks of the consumption of too
much sugar are e.g. adiposity, diabetes or caries. Hence,
sweeteners became popular, which give the desired taste
without causing the unwanted side-effects. Sweeteners
like cyclamates taste several times sweeter than sucrose
and thus only a tiny fraction of them is needed.
Furthermore, they cannot be metabolized, neither by the
human body nor by bacteria causing tooth decay.
As a consequence of its significance, sugars and nutrition
based on sugar-containing food are subjects in School. In
Primary School, pupils are usually only advised to reduce
the intake of food containing added sugar or even avoid it
at all. [1] A scientific inquiry approach to the subject in
Primary Schools is unknown to the author. In contrast,
descriptions of experiments in which pupils even in Primary
School perform experiments regarding starch in food are
well known. [2] Later on in High School or even
structures of simple mono- and disaccharides are typical topics.
[3] A characteristic property of many sugars which is usually
tested for is their behaviour to act as reducing agents. A
sugar is reducing, if its molecules contain an anomeric
carbon atom that is not involved in a glycosidic bond.
Fehling’s or Benedict’s test solutions as well as Tollens’
reagent are widespread and commonly used to test for
reducing sugars. Despite their frequent use, they have a
few drawbacks that will be outlined first.
2. Drawbacks of Fehling’s, Benedict’s and
Tollens’ Test
2.1. Necessity to Heat the Solution
None of the three tests for reducing sugars will work
quickly enough at room temperature for the purpose of a
chemistry lesson. Only with fructose, the typical olive
green precipitate occurred under this condition within
a minute. Heating with a bath of hot water is the
method-of-choice here, but the material therefore has to be
provided by the teacher. Given the same temperature and
concentration of sugar, Fehling’s test proceeds quicker
than Benedict’s, which is due to the higher concentration
of hydroxide ions in the former.
46 World Journal of Chemical Education
2.2. Chemical Hazards and Disposal
Each chemistry teacher has to deal with two questions
when planning an experimental lab course for the students:
Can the handling of the chemicals used by the students be
in any way risky for them? And how are the chemicals
properly disposed?
All three reagents consist of strongly alkaline solution
of heavy metal compounds. According to the Globally
Harmonized System (GHS), they are classified as toxic if
swallowed and irritating to skin and eye. Since they are
very toxic to aquatic life, the must not be drained in the
sink but have to be collected and neutralized. According to
those properties, the above mentioned teacher may want
to consider an alternative for Fehling’s, Benedict’s or
Tollens’ reagent in order to reduce the endangering and to
safe costs for disposal.
2.3. Interpretation of Fehling’s and
Benedict’s Test
Fehling’s and Benedict’s test for reducing sugars are
frequently misinterpreted. [4,5] It was shown as early as in
1927, that glucose is not oxidized to gluconate or to
gluconic acid, respectively, by alkaline solutions of
copper(II)compounds. [6] In the following year, EVANS
and co-workers investigated the reaction at room temperature
in a weakly acidic solution. They identified glucosone i.e.
2-keto-glucose, as the primary product of the reaction. [7]
It is not stable and subsequent cleavage of the C1−C2 bond
is a first step of a series of fragmentation reactions. The
spectrum of products seems to be rather sensitive towards
temperature, pH and complexing agents. [8] It is usually
assumed that Fehling’, Benedict’s and Tollens’ reagents
oxidize simple aldehydes to the corresponding carboxylates.
Based on this assumption, the wrong conclusion for glucose
is drawn, leading to the above mentioned misinterpretation.
For Fehling’s and Benedict’s test, this assumption was
experimentally checked and could not be confirmed. [9]
As far as Tollens’ reagent is concerned, neither the reaction
product with glucose nor that with simple aldehydes has
been published, to the best knowledge of the author. Thus,
Fehling’s and Benedict’s test can be used to unequivocally
identify reducing sugars. The chemistry of these reactions
is but rather complex and can hardly be dealt with in detail
even at the High School level.
3. The Iodine Test
First investigations of the oxidation of glucose by
iodine in alkaline solutions date from the early 20th
century. [10,11] Their purpose was a quantitative analyses
Figure 1. Oxidation of D-gluconopyranose to D-δ-gluconolactone by hypoiodous acid
of glucose in natural sources. Different from the reaction
with Fehling’s and Benedict’s test solution, glucopyranose
is oxidised to δ-gluconolactone (see Figure 1). The latter
is hydrolysed to gluconate under alkaline conditions.
[12,13,14].
The presence of iodine in a weakly alkaline solution
can be indicated by adding a few drops of a diluted
solution of amylose (“starch”), leading to the typical blue
colour. The colour disappears, if glucose is added to the
solution.
The active oxidizing agent in the reaction is not iodine
itself but hypoiodous acid, HOI. The latter is formed from
iodine in reaction (1): [12,13]
I2 + OH −  I− + HOI (1)
An investigation of the reaction rate at different pH
values by INGLES und ISRAEL supported this hypothesis.
The authors obtained a maximum rate at a pH close to pKa
of HOI (11,0), where c(HOI) has its maximum as well. At
higher pH, c(HOI) is lowered by reaction (2) and (3). The
iodate ion formed in (3) does not oxidise glucopyranose.
HOI + OH −  OI− +H 2 O (2)
3HOI+3OH −  2I− +IO3− +3H 2 O (3)
At lower pH-values equilibrium (1) is shifted to the left
side and thus c(HOI) is lowered as well. In order to keep
the pH-value in a narrow range close to the pH for a
maximum reaction rate, an appropriate buffer solution
should be used.
For the purpose of qualitative analyses, three different
solutions are needed in order to perform an iodine test.
The concentrations of iodine, sodium bicarbonate and
sodium carbonate can be varied, the values given here are
those used for the present work.
 Solution A: Iodine (c0(I2) = 0,001 mol·L−1)
dissolved in an aqueous solution of potassium
iodide (c0(KI) = 0,01 mol·L−1).
 Solution B: Buffer solution of sodium carbonate
(c0(Na2CO3) = 0,25 mol·L−1) and sodium
bicarbonate (c0(NaHCO3) = 0,25 mol·L−1) in water.
 Solution C: Solution of soluble starch in water,
w(starch) = 0,1 %.
The iodine test is positive not only for reducing sugars.
Several other compounds react with the iodine or the
hypoiodous acid. The most prominent are given here:
 Ascorbic acid
 Ethanol
 Acetone and other ketones whose molecules contain
C−H bonds at an α-carbon atom.
 Unsaturated compounds, especially unsaturated fatty
acids.
 World Journal of Chemical Education
4. Pedagogical and Methodological
Objectives
The reaction of glucose and also of other reducing
sugars with a weakly alkaline solution of iodine is a
versatile tool, offering a variety of useful applications to
chemistry in School classes and even undergraduate
University courses. Its main advantages over Fehling’s
and Benedict’s test are:
 No solutions with extreme pH or such containing
heavy metal compounds are necessary. If direct
contact with the eyes is avoided and it is not
swallowed, the iodine test solution is harmless.
 The reaction proceeds rapid enough even at room
temperature, so no additional heating is necessary.
Hence, no glassware is needed. Simple small beakers
made of plastics are suitable reaction vessels.
It is these two properties, which render the iodine test
suitable even for a use in Primary School, where different
foods can be tested for reducing sugars (see 4.1).
At the High School level, reducing and non-reducing
sugars can be distinguished. The reaction, following a
simple and straightforward stoichiometry (see Figure 1),
can be understood at that level. Also, hydrolysis of the
glycosidic bond in sucrose can be investigated. As well,
the increase of the rate of a chemical reaction with increasing
concentration of temperature can be easily deduced.
Finally, the positive iodine test of a solution of fructose, a
ketose, and its reduced rate can be rationalized in terms of
the DE BRUYN-VAN EKENSTEIN-rearrangement. [15]
4.1. The Iodine Test in Primary School
According to Piaget’s theory of cognitive development,
pupils in Primary School are in the so called concrete
operational stage. [16] They develop logical thinking,
Figure 2. The iodine test with chewing gum containing sugar (A) and without sugar (B). Left: just after chewing gums were added. Right: after 5 minutes
Figure 3. The iodine test with table sugar (left), grape sugar (middle) and grape juice, one minute after adding them to the iodine test solution.
47
which is a prerequisite for every scientific enquiry.
Regarding the enquiries presented, 4th to 6th year Primary
School kids should be capable to understand a logical
conclusion of the following, formal type: If A ⇒ B,
then ⌐B ⇒ ⌐A. A is “Solution X contains grape sugar”. B
is “A weakly alkaline solution of iodine and starch that is
added to solution X, becomes colourless.” Hence, the kids
should be able to tell, if a solution does not contain grape
sugar. It might be a challenge to avoid that pupils
conclude A from B. I.e. they might regard a positive
iodine test as a proof for the presence of grape sugar. In
that case, the iodine test with ascorbic acid or malt sugar
can demonstrate, that other compounds can be the cause
for decolourisation as well. Two experiments are proposed
for the above mentioned purpose.
Experiment 1: A problem to start with could be the
following “Some chewing gums are good for keeping teeth
healthy but others are said to be harmful. The healthy ones
are free of sugar, but all chewing gums taste sweet. How
can we distinguish between them without tasting?” The
iodine test has to be presented and Figure 2 shows the
results. Experimental details are given in Chapter 5.
The kids are instructed to observe the different
behaviour of a chewing gum containing sugar compared to
the sugar-free chewing gum. It should become clear to
them, that only the sugar containing chewing-gum will
lead to a decolourization of the iodine test solution. In a
second stage, they should use the test to experimentally
recognize sugar-containing chewing gum.
Experiment 2: Is it table sugar that gives ripe grapes a
sweet taste? The iodine test allows a distinction between
table sugar and grape sugar. The former is a non-reducing
sugar, i.e. the test-solution will stay blue when table sugar
is added to it. In contrast, if grape juice from ripe grapes is
added, the blue colour will rapidly disappear. The same
happens, if grape sugar is added.
48 World Journal of Chemical Education

The kids should deduce from their observations that the
sweet taste of the grapes cannot be caused by table sugar
only. Instead, that is what they have to be told, grapes
contain grape sugar. At this stage, kids can come up with
the conclusion, that chewing gums cannot contain only
table-sugar.
4.2. The iodine test in High School
Students at High School should be able to master a
more abstract view on the chemistry of simple
carbohydrates. Given the name of the compound, they are
supposed to deduce the correct Fischer or Haworth
formula (Figure 4) of the mono- and disaccharides.
Subsequently, they have to decide by the criterion of a
present hemiacetal group, whether it is a reducing or a
non-reducing carbohydrate.
In a simplified way, the reaction of a reducing sugar
with the iodine test solution (see Figure 1) could be
presented with the iodine molecule as the oxidizing agent,
the hydroxide ion acting just as proton acceptor.
Experiment 3: Reducing and non-reducing sugars. The
four carbohydrates given in Figure 4 are tested for their
reducing action. 1, 2 and 3 will lead to a rather quick
decolourization of the iodine test solution, as forecasted
on the basis of the molecular structures. 4 does not change
the blue colour of the solution, since sucrose is a non-
reducing sugar.

Figure 4. Haworth formula of selected monosaccharides (1 and 2) and disaccharides (3 and 4). (1: β-D-glucopyranose; 2: α-D-galactopyranose; 3:
maltose; 4: sucrose. Hemiacetal-groups are marked)

Figure 5. Simplified reaction equation for the oxidation of reducing sugars D-glucopyranose by the iodine test solution

Figure 6. Mixtures of the iodine test solution with carbohydrates 1 – 4. Left: Immediately after addition to iodine test solution. Right: 30 seconds after
mixing of the solutions
 World Journal of Chemical Education 49

Experiment 4: Lemonade – does it really contain sucrose?
Lemonade tastes sweet because sucrose was added during
the production process. Students know sucrose as a
non-reducing carbohydrate and should thus be surprise
that lemonade gives a positive iodine test. Of course,
lemonade contains other ingredients as well. Most of them
contain flavouring and colouring compounds and citric
acid, some may contain ascorbic acid. The latter is known
to reduce iodine to iodide – but this reaction proceeds
already quick in neutral or weakly acidic solutions.
Reducing sugars and ascorbic acid could hence be
distinguished by reaction with iodine test solution at
different pH, removing the latter first by this reaction.
At this stage, students should become familiar with the
hydrolysis of the glycosidic bond, a reaction catalysed by
protons (see Figure 7).
The equilibrium of this reaction is shifted towards the
right side. Since lemonades are acidic solutions, they
contain a suitably high concentration of protons. Hence, if
sucrose was added in the production stage of the lemonade,
it has been hydrolysed to give glucose and fructose.
Mainly glucose is responsible for the positive iodine test.
Fructose has to isomerize to glucose before (see
Experiment 7), so its contribution is only of minor
importance. Since the acid in the lemonade lowers the pH
of the iodine test solution, only little lemonade should be
used. As well, a 0,25 molar solution of sodium carbonat
can be substituted for the buffer solution.

Figure 7. Hydrolytic cleavage of the glycosidic bond in the sucrose molecule, leading to the formation of glucose and fructose

Figure 8. The iodine test with regular lemonade (A) and sugar-free lemonade (B). Left: just after lemonade was added. Right: after 4 minutes

Figure 9. Alkaline iodine-starch-solutions to which equal volume of glucose solutions with different concentrations were added. The pictures show
different stages of the reaction (times as indicated). (1) c(glu) = 0,5 mol·L−1; (2) c(glu) = 0,2 mol·L−1; (3) c(glu) = 0,1 mol·L−1, (4) c(glu) = 0,05 mol·L−1
50 World Journal of Chemical Education
“Sugar-free lemonade” can be tested, too, and a
comparison to lemonade will give a similar result as for
the chewing gums (see Figure 8 and Figure 2).
Experiment 5: Concentration and the rate of reaction.
The rate at which the oxidation of the reducing sugar proceeds
can be qualitatively estimated by the time it takes the
colour of the iodine-starch-solution to disappear. [17]
(see Figure 9) Thus, apart from being a probe for reducing
sugars, the iodine test can help to understand factors ruling
the rate of a reaction. A higher concentration of glucose
leads to a quicker disappearance of the colour of the
iodine-starch-solution. If c0(I2) is the starting concentration of
iodine in the test solution and ∆t is the time it takes the
solution to become colourless, ∆c0(I2)/∆t is a measure for
the reaction rate. c0(I2) being held constant, the rate increases,
if ∆t becomes smaller. The observations made can be
rationalized in terms of a simple collision theory with respect
to the concentration of glucose. The more glucose molecules
are in the solution, the higher is the probability for a reactive
collision with a molecule of hypoiodous acid (or iodine in
a simpler view of the reaction). Nevertheless, ∆t as defined
above cannot be measured objectively. In order to obtain
reliable values for the rate of the reaction, the course of
the reaction has to be followed by photometry. [17]
Figure 10. Alkaline iodine-starch-glucose-solutions of equal concentrations at different temperatures. (times as indicated). (1) T = 293 K, (2) T = 303 K,
(3) T = 313 K
-
OH H O
H OH
O O
HO H + OH- HO H (2)
H OH H OH
H OH - H2O H OH
OH OH
(1) (4)
Figure 11. DE BRUYN-VAN EKENSTEIN-rearrangement between fructose (1), glucose (2) and mannose (3) via the common endiolate-form (4)
Experiment 6: Temperature and the rate of reaction.
Regarding the influence of temperature, a straightforward
observation is the time ∆t becoming shorter with
increasing temperature. An increase in temperature
of 10 K reduced the time needed for the colour to
disappear approximately to one forth. (see Figure 10).
The exponential temperature dependence found can
be understood in terms of the Arrhenius equation:
k(T) = A·exp(−EA/R·T), where k(T) is the rate
temperature dependent rate constant, A is the collision
factor, EA is the molar activation energy, R is the gas
constant and T the absolute temperature.
Measuring the rate constant at different temperatures
allows an estimation of the energy of activation. Details of
this approach will be given in another paper. [17]
Experiment 7: If fructose is added to a weakly alkaline
solution of iodine and starch, the blue colour of the
solution disappears as well – much slower though than
with glucose (see Figure 12). This is a surprising
observation, since fructose is a ketose and cannot form
a hemiacetal that can be oxidised to a lactone. The
positive iodine test and its reduced rate can be rationalized
in terms of the DE BRUYN-VAN EKENSTEIN-rearrangement
(see Figure 11). [15]
H O
H OH
HO H
H OH
+ OH- H OH
- H2O
+ OH-
H O
- H2O HO H
HO H
H OH
H OH
OH
(3)
 World Journal of Chemical Education
Figure 12. Comparison between equally concentrated solutions of D-glucose (1) and D-fructose (2) with respect to the iodine test. Times as indicated
In alkaline solution, fructose (1) can be transformed
either to glucose (2) or mannose (3), the reaction
proceeding via the endiolate ion (4). Hence aldoses are
produced, which subsequently can be oxidised by the
hypoiodous acid. A comparison of D-glucose and D-
fructose demonstrates, that this transformation proceeds
rather slow (see Figure 12).
5. Experimental
5.1. Preparation of Solutions
The three solutions necessary for the iodine-test,
subsequently named A, B and C, were prepared as follows:
Solution A: Aqueous solution of iodine (c(I2) = 0,001
mol·L−1) and potassium iodide (c(KI) = 0,01 mol·L−1).
Iodine, I2, (m = 0,254 g) and potassium iodide, KI, (m =
1,660 g) were dissolved in demineralised water in a sealed
Erlenmeyer flask. The solution was then filled up to V =
1000 mL in a standard measuring flask.
Solution B: Buffer solution of sodium carbonate
(c(Na2CO3) = 0,25 mol·L−1) and sodium bicarbonate
(c(NaHCO3) = 0,25 mol·L−1). Anhydrous sodium
carbonate, Na2CO3 (m = 26,50 g), and sodium bicarbonate,
NaHCO3 (m = 21,00 g) were dissolved in demineralised
water. The solution was then filled up to V = 1000 mL in
a measuring flask.
Solution C: Starch solution (w(starch) = 0,1 %). In a
beaker, water was added to soluble starch (m = 0,1 g) until
the mass of the mixture summed up to 100,0 g. The
mixture was heated in a microwave oven until the starch
dissolved. The hot solution was filled in a 100 mL
dropping bottle, sealed and cooled in a water bath.
Solutions of D-glucose (experiments 5 to 7) and
D-fructose (experiment 7), were prepared as follows:
Solution of D-glucose (c = 0,5 mol·L−1). D-glucose-
monohydrate, C6H12O6·H2O (m = 9,90 g) was dissolved in
water. The solution was then filled up to V = 100 mL in a
measuring flask.
Solution of D-glucose (c = 0,2 mol·L−1). 40 mL of
the glucose-solution (c = 0,5 mol·L−1) were filled up to
V = 100 mL in a measuring flask.
Solution of D-glucose (c = 0,1 mol·L−1). 50 mL of
the glucose-solution (c = 0,2 mol·L−1) were filled up to
V = 100 mL in a measuring flask.
Solution of D-glucose (c = 0,05 mol·L−1). 50 mL
of the glucose-solution (c = 0,1 mol·L−1) were filled up to
V = 100 mL in a measuring flask.
Solution of D-fructose (c = 0,5 mol·L−1). D-Fructose,
C6H12O6 (m = 9,00 g) was dissolved in water. The solution
was then filled up to V = 100 mL in a measuring flask.
51
5.2. Experimental Procedures
General procedure: If not stated otherwise, for each test,
1,0 mL of each of the solutions A, B and C were mixed in
a small plastic beaker (20 mL).
Experiment 1: Two beakers with test solutions were
prepared. To one of the solutions, a slice of approximately
1 cm length cut from a sugar-containing chewing gum was
added. An equally sized slice of a sugar-free chewing gum
was added to the other solution. The mixtures were
steadily tossed carefully and observed for changes in
colour during five minutes.
Experiment 2: Three beakers with test solutions were
prepared. A grape is cut in half. The two halves are
squeezed between two spoons and the juice thus release is
collected in a forth beaker. Pea-sized amounts of table
sugar (sucrose) and grape sugar (glucose), respectively,
are added to two of the beakers. They are at least partially
dissolved by slightly tossing the beakers. The grape juice
is added to the third beaker. The mixtures were observed
for changes in colour occurring within a few seconds.
Experiment 3: Four beakers with test solutions were
prepared. Pea-sized amounts of D-glucose (1), D-galactose
(2), maltose (3) and sucrose (4) were added. The solids are
at least partially dissolved by slightly tossing the beakers.
The mixtures were observed for changes in colour
occurring within a few seconds.
Experiment 4: Since lemonade is an acid solution, the
volume of the buffer solution here is 2 mL instead of 1
mL for each test solution. Two beakers with test solutions
were prepared. Five drops of “regular”, sugar containing
lemonade and sugar-free lemonade, respectively, are
added. The mixtures were observed for changes in colour
occurring within a few minutes.
Experiment 5: Four beakers with test solutions were
prepared. Then, simultaneously 1,0 mL of D-glucose-
solutions with concentrations 0,5 mol·L−1, 0,2 mol·L−1,
0,1 mol·L−1 and 0,05 mol·L−1 were added. The time, when
the colour seems to disappear was measured.
Experiment 6: Three test tubes were filled with the
iodine-test solution. Three more test tubes were filled
with a solution of D-glucose (c = 0,5 mol·L−1). Their
temperatures were set to 293 K, 303 K and 313 K,
respectively, by putting them for five minutes into
appropriate water baths and shaking the tubes occasionally.
1,0 mL of the D-glucose-solution was simultaneously
added to the test-solution with the same temperature. The
time, when the colour seems to disappear was measured.
Experiment 7: Two beakers with test solutions were
prepared. Then, simultaneously 1,0 mL of D-glucose- and
D-fructose-solutions with concentrations of 0,5 mol·L−1,
were added. The time, when the colour of either seems to
disappear was measured.
52 World Journal of Chemical Education

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